EP1981333A2 - Animal model for hiv induced disease - Google Patents

Abstract

HIV does not cause disease in any non-human species. Thus, there is no animal model system to evaluate the efficacy of strategies aimed at preventing, treating or curing disease caused by this virus. The present invention provides compositions and a method for producing an animal model for HIV induced disease. The present invention is an animal adapted to simulate a human-like immune response to HIV, which is accomplished by activation and inactivation of complement of proteins within the animal. Accordingly, the present invention stages certain human proteins within an animal by way of its gut associated lymphoid tissue followed by infection of live HIV.

Description

ANIMAL MODEL FOR HIV INDUCED DISEASE

RELATED APPLICATION DATA

[0001] This application claims priority to U.S. provisional application No. 60/765,315, filed on February 3, 2006, which is hereby incorporated by reference.

TECHNICAL FIELD

[0002] The present invention relates to a composition and method for producing an animal model for HIV.

BACKGROUND ART

[0003] HIV is a viral infection. Therefore, by definition, HIV is an intra cellular parasite. The virus must assimilate a variety of host cellular proteins, lipids, carbohydrates and nucleic acids into its own structure and reproductive cycle. Attempts at inoculating animals with HIV have all failed. Animals such as mice lack one or more cellular proteins or other cellular derived molecules necessary for viral replication, immune evasion and immune suppression. The purpose of this invention is to produce an animal that possesses the full complement of HIV immune mediated molecules in a manner that the animal can assimilate in trans the human derived proteins necessary for an HIV infection to proliferate. The animal will not recognize these foreign molecules as being foreign, and therefore, will not develop an immune response towards them. Furthermore, these human derived molecules will be directed towards Peyei^s patches, the very site of HIV replication. The animal will be susceptible to HIV disease.

[0004] Rationale behind an HIV animal model

1. Allow for an in depth and ethical study of the natural course of HIV infection. Currently all studies are on human subjects and are therefore limited by ethical considerations.

2. A testing ground for anti-retroviral drugs and other technology.

3. A testing ground for HIV based vaccines.

4. Allow the development and manufacture of effective HIV vaccines. In 1794, Edward Jenner demonstrated that inoculation of humans with extracts from cowpox lesions produce minimal systemic disease but protected the recipient from smallpox. Initially, the only way to supply the population with enough cowpox vaccine was to pass the infection (cowpox) from person to person by serial infection. This methodology, however, was complicated by transmission of other diseases such as syphilis and hepatitis and fell into disfavor. The cowpox vaccine was later passed into sheep and water buffalos in an attempt to obtain enough inoculum for the population. Recently, an unused smallpox vaccine was uncovered in New York dating back to 1876. This virus was identified as vaccinia. By 1876, the original cowpox vaccine was replaced by vaccinia virus. Vaccinia is not found in any animal studied to date. It most likely resulted as a recombination of cowpox with other pox vectors, animal and human. The U.S. Smallpox Vaccine (Dryvax by Wyeth) reserve is over thirty years old and was derived from a seed stock of a New York City Board of Health strain that was passed between twenty-two to twenty- eight times on young calves. Distribution of Dryvax ceased in 1983. Multiple retroviral vectors infect animals. Passage of HIV and one or more animal retroviruses will allow for multiple recombinant events to occur. In a manner that parallels the vaccinia vaccine derivation an HIV vaccine can be developed. Such an animal model can also be a continuous inexpensive reliable source of new fresh vaccine.

Overview of HIV Lifecycle and Protein Requirements

[0005] A retroviral life cycle can be divided into an afferent and efferent limb. The afferent limb starts with viral attachment and ends with viral DNA integration into the host genome. The efferent limb commences with the production of viral messenger RNA and culminates with viral fission releasing immature virions. [0006] The afferent lifecycle of the virus will be arbitrarily divided into the following steps: 1. Attachment to a target cell by its surface (SU) and transmembrane (TM) proteins. The surface protein binds to the CD4 receptor and to either the CCR5 or CXCR4 coreceptor.

2. Fusion of viral envelope and cell plasma membrane.

3. Deposition of viral core into cytoplasm.

4. Reverse transcription of viral RNA.

5. Translocation of viral pre-integration complex across nuclear membrane.

6. Integration of viral DNA into host DNA.

[0007] The efferent lifecycle of the virus will be arbitrarily divided into the following steps:

7. Transcription of viral RNA into RNA.

8. Splicing of viral RNA.

9. Translocation of early viral completely spliced RNA products (Tat, Rev and Nef) across nuclear membrane into cytoplasm.

10. Rev mediated translocation of singly spliced and unspliced viral RNA across the nuclear membrane to the cytoplasm.

11. Viral env proteins produced in cytoplasmic rough endoplasmic reticulum (RER).

12. Glycosylation and folding of env proteins in Golgi apparatus. 13. Targeting of mature envelope proteins to cytoplasmic side of plasma membrane.

14. Translation of Gag and Gag-Pol polyprotein.

15. Targeting of Gag and Gag-Pol polyprotein to host endosomal machinery.

16. Gag and Gag-Pol polyprotein cleaved by viral protease.

17. Assembly of Gag and Gag-Pol polyprotein precursors and envelope proteins at budding site.

18. Viral fission.

19. Viral maturation.

[0008] Each step delineated above relies on host derived proteins, lipids, carbohydrates and/or nucleic acids. Animals do not support the HIV lifecycle because they lack one or more necessary host derived molecules.

[0009] HIV, as with all significant viral pathogens, is able to evade the host immune response. Furthermore, HIV down regulates or deregulates the host immunologic response.

[0010] For an animal model to be successful for HIV disease, three correlates of the disease must be expressed: 1. Viral replication 2. Viral immune evasion

3. Viral immune deregulation and/or suppression

[0011] Many proteins necessary for viral replication of the host immune response are human host derived proteins that are not found in animals. These include, but are not limited to, tRNA synthetase, tRNA^lys, Tsg101 , TaI, Staufen, LEDGF/p75, Cyclin T, CDK9 and RNA polymerase II. To create an animal model capable of not only supporting HIV replication, but also reproducing HIV disease in the animal requires the assimilation of these proteins into the animal without the animal recognizing these proteins as foreign. Success of such an animal model would rely on the lack of an immunologic response to these human proteins. Furthermore, assimilation or targeting of these proteins into the proper target tissues, predominantly Peyei^s patches, the principal site of HIV replication, is necessary to reproduce an HIV infection in an alternate host.

[0012] Viral evasion of the host's immune response requires the active participation of host derived cellular proteins such as the complement control proteins CD55, CD46 and Factor H. These proteins are necessary to prevent the host's immune cells from reacting to and destroying normal tissue. By incorporating these molecules into an intact HIV virion, the virus is able to fool the immune system in a "cloak-and-dagger" method that avoids virolysis. [0013] Immune disregulation is accomplished by the virus skewing the host towards a Th2 immune response. This is accomplished by the virus hijacking the endosomal pathway by incorporating molecules such as Tsg101 , TaI and Ubiquitin. Furthermore, the viral envelope incorporates MHC-II and CD86 molecules which are consistent with a Th2 response.

[0014] As a corollary to the above paragraph, any given protein may exhibit different and at times divergent and conflicting functions, complicating the challenge to an animal model for HIV.

DISCLOSURE OF THE INVENTION

[0015] The present invention provides compositions and a method for producing an animal model for HIV induced disease. The present invention is an animal adapted to simulate a human-like immune response to HIV, which is accomplished by activation and inactivation of complement of proteins within the animal. Accordingly, the present invention stages certain human proteins within an animal by way of its gut associated lymphoid tissue followed by infection of live HIV.

BEST MODE(S) FOR CARRYING OUT THE INVENTION [0016] The present invention is directed to an animal model for HIV and the method of producing the same. Preferably, the present invention is a mouse adapted to simulate a human-like immune response to HIV, which is generated by appropriate protein behavior within the mouse. The mouse genome has been published.¹ Extensive linkage conservation/synteny between mouse and human DNA has been established.² The present invention stages certain human proteins within a mouse by way of its gut associated lymphoid tissue (GALT).

[0017] A key to protein variability lies in the primary, secondary, tertiary and quaternary structure of the protein itself. The protein may assume different secondary, tertiary and quaternary structures in various environmental conditions. Changes in ph, temperature, as well as the presence, absence, or concentration of cellular cofactors, such as calcium and magnesium, alter the structure and function of the protein. Most importantly however, proteins can be divided into basic building blocks or subunits known as motifs, each which possesses a specific function which is independent of the rest of the molecule. In some instances only a portion of the protein is directly involved in a certain metabolic process. The whole protein may or may not be needed to produce the desired effect. The subunits not directly involved in the cellular activity may affect the overall structure, stability, intracellular location and often function as a scaffold. [0018] However it has also been demonstrated in other circumstances that a subunit of a protein that carries a significant function maintains that function when physically separated from the rest of the molecule. In such circumstances one may envision that only a portion of the protein is needed to perform the desired effect and is necessary to be encoded by recombinant DNA technology to develop an animal model for HIV. Invariant amino acids in each protein are always noted. For example, the cystine residue occupying the position of amino acid 261 of Cyclin T is absolutely required for interaction with Tat.³

[0019] The above conclusion has been demonstrated with in vitro models of human CyclinTI (hCycTI ) as it interacts with the Tat protein. A heterodimer of human CyclinTI and Tat protein is a prerequisite to the binding of the heterodimer to the TAR sequence that initiates HIV RNA replication. The first 272 amino acids of the 726-aa hCycTI protein are sufficient to support Tat function, TAR recognition and binding and ultimately viral replication. Even more specifically a critically defined region of hCycTI located between residues 250 and 262 is critical for Tat and TAR binding and has been termed the Tat-TAR recognition motif (TRM).

[0020] All proteins have a characteristic half life usually measured in minutes or hours. Therefore, these proteins that support HIV replication and immune evasion need to be produced within the animal in a continuous pattern with a steady state level. The tissue concentration of the proteins supplied in trans should mirror that found in the normal human immunologic milieu.

[0021] All proteins administered to the animal model are encoded within the DNA. Recombinant technology allows introduction of human DNA into bacteria, fungi, yeast or viruses. Utilizing commensal organisms, found normally in the gut of an animal such as a mouse, rat or rabbit for this recombination the proteins of human origin necessary for HIV replication and immune evasion and immune disregulation can be introduced into the animal without the animal rejecting the proteins as foreign. The mechanisms of suppressor cells and regulatory cells found within the gut associated lymphoid tissue (GALT) prevent immunologic response to ingested food, commensal organisms and the products of the commensal organisms. Commensal organisms often produce vitamins necessary for the host to survive. Vitamins are protein based structures. By reasonable inference other proteins produced by the commensals would be assimilated into the host without an ensuing immunologic response. To replicate and survive the commensal bacteria continually produce protein and other components of its structure in excess of what is needed or incorporated into the replicating bacteria. These excess proteins do not elicit an immunologic response from the host animal.

[0022] GALT constitutes nearly 80% of the total body's immune cell population. GALT is the most comprehensive lymphoid organ system in humans. The function of GALT is a paradox and at times is in conflict with the systemic immune system. The systemic immune apparatus, under normal conditions, functions in a sterile environment devoid of pathogens and pathogen associated toxins. Therefore, any foreign matter encountered by the systemic immune system is regarded as a potentially harmful invader and the appropriate immunologic response follows. GALT₁ however, stands as a barrier between the human organism and an external environment replete with foreign tissue. The foreign matter includes a variety of commensal organisms, commensal derived products, pathogens, and pathogen derived products and ingested food. The entire Gl tract from the mouth to the anus is functionally external to the human body. Unlike the systemic immune system, which responds vigorously to any foreign matter, GALT must differentiate between commensal organisms and their products, as well as ingested food to which an immunologic response would have adverse consequences and invading pathogens potentially lethal to the host.⁴

[0023] To affect this diversity of function, GALT is compartmentalized and, in contrast to the systemic and peripheral immune system (spleen & lymph nodes), is characterized by non-homogeneously distributed B and T cells. The phenotypic behavior, cell surface markers, developmental origins, secretory products, and hence function of the T and B cells of GALT, is markedly different from the T and B cells of the systemic system. Furthermore, GALT contains certain subsets of non-conventional lymphocytes such as γ/δ T cells. Overall GALT is characterized by afferent and efferent conduits not found in the systemic system.⁵

[0024] GALT (armed with a variety of immunologic cells not found in the systemic circulation, and patterned or clustered into characteristic vehicles not found elsewhere in the body) is capable of immunologic suppression as well as classically based Th-1 and Th-2 immune responses. Antigen uptake in GALT occurs through specialized epithelial cells known as "M" cells or "membranous" cells. Antigen uptake in GALT can also occur directly by epithelial cells in close proximity to underlying T and B cells. The uptake or assimilation of antigens through the "M" cells or epithelial cells may result in localized immune response, disseminated immune response and/or tolerance or immunosuppression. The vast majority of antigens interacting with GALT results in specific suppression of immunity for that antigenic structure. This is necessary because the primary function of GALT is to prevent an immunologic reaction to innocuous, and at times beneficial, foreign material.⁶

[0025] The final determination in GALT of immunity versus tolerance rests on many variables. These include but are not limited to the chemical structure of the antigen, the dose of the antigen administered, and the cytokine environment. Whether this phenomenon is termed suppression, anergy, deletion, ignorance, and/or immunologic deviation is irrelevant. Importantly, immunologic tolerance within GALT depends on an intact epithelial barrier.⁷

[0026] Many mechanisms have been described in the literature detailing the immune suppression observed with antigens derived from the large and small intestine. In classic immunology dendritic cells exposed to peripherally derived antigen assimilate the antigen (by a variety of mechanisms including but not limited to endocytosis, macrocytosis, pinocytosis, and cross presentation). Dendritic cells (DCs) lining the tissue have been described. The DCs then undergo a process of maturation and migrate to the most proximal lymph nodes. Expressing a "danger signal" the cells of the lymph node respond and eliminate the antigen expressed by the DCs. Recently however, DCs lining the GALT with an opposite function, one of tolerance have been described in the literature. These cells stimulate a protective immune response when stimulated by pathogens whose tropism (i.e., the phenomena observed in living organisms of moving towards each other) is confined to pathogens that infect or are confined to epithelial cells.⁸

[0027] The incorporation of the DNA encoding these human derived proteins into the commensals, herein referred to as incorporated DNA, can be done through recombinant technology with the following seven methodologies commonly used and known by those in the art.

1. Incorporation of the DNA into the bacterial, viral, yeast or fungal DNA utilizing restriction enzymes, endonucleases, exonucleases, deoxyribonucleases, ribonucleases, alkaline phosphatases, polynucleotide kinases, terminal transferases, and DNA ligases, all commercially available.⁹

2. The formation of a plasmid encoding the human protein. A plasmid is a genetic particle physically separate from the chromosomal DNA of the host cell that is stable and can function and replicate independently of the nucleus.¹⁰

3. Incorporation of the DNA into a bacteriophage. A bacteriophage is a virus with a specific affinity for bacteria and has been found in association with essentially all groups of bacteria. Like other viruses, they contain either RNA or DNA but never both.¹¹

4. Hybrid plasmid/phage vectors such as cosmids, phagemids or phasmids.¹²

5. Bacterial artificial chromosomes.¹³

6. Yeast artificial chromosomes.¹⁴

7. A combination of the above.

[0028] If incorporated into a plasmid, a promoter/regulatory region controlling the plasmid activity would need to be included. The assimilation of the protein produced by the commensal into the animal may occur by passive (ATP independent) or active (ATP dependent) means. The DNA encoding a cell penetrating peptide (CPP) may be fused with the DNA encoding the human protein(s) prior to the recombinant process incorporating the DNA into the bacteria. Many cell penetrating peptides have been defined in the literature and have been used to carry cargos (attached protein, carbohydrate or lipid molecules) into cells which would normally be impermeable to these attached structures. Cell penetrating peptides can pass through cell walls, nuclear membranes, as well as the membranes enclosing other intracellular organelles with ease.¹⁵

[0029] Alternatively, the DNA encoding the below mentioned human proteins necessary for HIV viral replication, immune evasion and immune disregulation can be spliced into the DNA of an animal. Intuitively this may seem to be the most logical answer. For some proteins such as the CD4 receptor and the CCR5 and CXCR4 co- receptor, this would be workable and perhaps preferable, since the proteins would be a component of the host cell plasma membrane. Many potential problems arise using that conceptualized framework for all the proteins. Most difficult would be the targeting of the needed proteins to the sites of HIV replication (i.e., Peyer"s patches). Furthermore, encoding a protein into the DNA of an organism does not equate to transcription and translation of the DNA and protein production. 70% of the DNA in a mammal is not transcribed and has been termed "junk DNA". Production of a transgenic or chimeric animal does not equate to tissue targeting. External control of animals genetically modified at the level of embryonic cells is problematic.

[0030] These issues may be addressed as the science relating to models progresses. However, the present invention, as a first conceptualized model, involves splicing the DNA for the needed human proteins into commensal organisms.

[0031] The host proteins necessary for HIV to attach to a target cell, penetrate the target cell and replicate within the target cell, include and are not limited to the following list. The following proteins, or the nucleotide sequences encoding these proteins, preferably should be included in a working animal model for HIV:

1. Transcription factors. a. NF_KB b. NFAT c. Sp1

2. Cellular cofactors. a. Cyclin T b. CDK9/PITALRE c. RNA polymerase Il d. Exportin 1/Crm1 e. Ran GTP f. Ran GTPase activating protein (RanGAP) g. Ran Binding Protein (RanBPI )

3. Cellular receptors. a. CD4

4. Cellular co receptors. a. CCR5 b. CXCR4 c. CCR2B d. CCR3 e. CCR8 f. GPR1 g. GPR15 (Bob) h. STRL33 (Bonzo) i. US28 j. CX3CR1 (V28) k. APJ

I. chemR23

5. Cellular proteases. a. Furin

6. Cellular proteins involved in the ubiquitin-proteasome pathway. a. H-β-TrCP b. Skpip 7. Cellular adaptor protein. a. AP-2

8. Human ribosomal RNA.

[0032] The host derived proteins necessary for HIV to evade the immune response include but are not limited to the following, and preferably should be included in a workable animal model for HIV. (See Table in Appendix A for a complete list of "Host Proteins Incorporated into the Intact Virus and /or Pre-1 ntegration Complex (PIC)".

1. Plasma proteins. a. C4 binding protein (C4b protein) b. Factor H ( Includes FHL-1 , FHR1 , FHR2, FHR3, FHR4, FHR5)

2. Cell membrane bound proteins. a. Membrane cofactor protein (MCP) or CD46 b. Decay accelerating factor (CD55) c. Complement-receptor 1 (CD35) d. Complement-receptor 2 (CD21 )

3. Homologous restriction factor (HRF).

[0033] Finally and in addition to the proteins listed above the table located in Appendix A lists the host proteins incorporated into the intact virus, the pre-integration complex (PIC) and those involved in the HIV lifecycle. It is not exhaustive as new viral protein/host protein interactions are reported in the literature with regularity. The genetic loci of the human proteins have been described in the literature and allow for restriction enzyme splicing into yeast, bacteria or plasmid DNA.

[0034] In an alternative embodiment, the activity of Human Factor H in an animal can be limited by administration of soluble complement-receptor 1 (sCR1 ) by adding sCR1 exogenously or by splicing the genomic sequence for sCR1 into a commensal organism. This protein binds to C3b and C4b and facilitates the breakdown of these proteins by Factor I. By binding to C3b, sCR1 prevents complement activation by the C3 convertase. The activity of Human Factor H in thwarting the complement cascade is mimicked by sCR1.

[0035] The administration of soluble CR1 is a controlled element or variable in the animal model. sCR1 allows control of tissue levels of C3b thereby limiting the activity of the C3 and C5 convertases which mirrors the function of Factor H.

[0036] In some animal models (e.g., old world primates), and particularly cell cultures derived thereof, TRIM-α confers a potent post entry (i.e., meaning after entry into the cell) block to HIV-1 infection. Cyclophilin A (CypA) binding to viral capsid proteins results in a similar response observed in vitro for certain human cell lines. Among new world primates, only owl monkeys exhibit post-entry restriction of HIV-1 replication. More specifically, monkey kidney cells of the Aotus trivirgatus owl restrict HIV infection, but are permissive for SIV infection. HIV restriction in these cells is completely abrogated when the interaction of the HIV-1 capsid and the cellular protein CypA Is disrupted. Paradoxically, the opposite is seen in human cells where capsid-CypA interaction is required for efficient intracellular HIV-1 replication. Therefore if such an animal model is used the viral capsid interaction with the host CypA protein must be severed. The use of the CypA-binding drug cyclosporine A (CsA) would be necessary if these animal models were used. Similar findings may exist in other animals but have not yet been delineated.¹⁶

[0037] The most effective weapon for immune perturbation within the HIV arsenal is the Tat protein. The Tat protein is necessary for viral replication as well. A multiplicity of immune down modulating effects of the Tat protein has been well documented in human studies. An accurate model of HIV must include Tat mediated immune suppression. This will involve the Tat protein and the host cell receptors for the Tat protein.

[0038] Expression of MHC class Il genes is inhibited by the Tat protein resulting in profound immunosuppression. A central protein in class Il expression is the class Il trans-activator (CIITA) protein. CIITA is responsible for integrating several proteins at the promoters of MHC class Il genes enhancing MHC Il gene transcription and ultimately MHC Il gene expression.

{0039] In human models, the Tat protein Inhibits CIITA function down regulating the expression of MHC Il genes. Human cyclin T1 (hCycTI) is involved in this Tat mediated immunosuppression.

[0040] In mice however, the Tat protein does not interact with the human counterpart of hCycTI , mouse cyclin T1 (mCycTI ). However, the Tat protein in mice does inhibit the activity of CIITA in a mechanism that is not dependent on mCycTI. The results are the same: the down regulation of the CIITA protein, decreased MHC Il production, and immunosuppression.

[0041] Co-expression of transfected human CD4, CCR5 and CXCR4 molecules into murine cell cultures allows entry of HIV-1 but replication is blocked. Murine cyclin T1 binds Tat but does not bind TAR. Transfection with human cyclin T1 restored Tat function.¹⁷

[0042] Murine cyclin T2 can bind HIV-1 Tat and facilitate TAR binding if a single residue, asparagine 260 is replaced with a cysteine residue. Interestingly, Tat from HIV-2 does bind murine cyclin T1 and murine cyclin T2. However, neither complex binds effectively the TAR residue. With both HIV-1 and HIV-2 Tat effective binding and activity of Tat on HIV replication is rescued in murine cells by the above-mentioned mutation of Cyclin T2 at amino acid number 260. Therefore, if a murine model is anticipated, mutation of Cyclin T2 at residue 260 would equate to human Cyclin T1 supplied in trans. In an alternate murine animal model, _oanother single amino acid difference between human Cyclin T1 and murine Cyclin T1 determines species restriction of HIV-1 Tat function. In this model, replacing the tyrosine residue at amino acid 261 in the murine Cyclin T1 with a cysteine conferred effective Cyclin T1 function with Tat and TAR.¹⁸

[0043] A competent Cyclin T1 is necessary but not sufficient for HIV viral replication.. This can be provided to a murine model by either one of the above-mentioned mutations in the mouse genome or by providing human Cyclin T1 in trans.

[0044] An effective block of HIV replication in a murine model is the inability of the virion to assimilate murine Factor H. HIV directly activates the classical complement pathway in rabbit, mouse and guinea pig serum. This activation results in viral neutralization by lysis.¹⁹ Factor H is bound at multiple sites to gp120 and gp41 in the intact virus.²⁰ Factor H is the main contributor to HIV evasion of complement mediated lysis.²¹ Murine and human Factor H is composed of twenty repetitive units and each unit is approximately sixty amino acids long.²² Neither murine Factor H nor human Factor H is characterized by an alpha helix or a beta pleated sheet. Both human and murine Factor H exists in two different conformational states (<pi and q>₂) that can be separated by hydrophobic chromatography. Both have equal function.²³ Although murine Factor H possesses a high degree of homology to human Factor H, it does not bind to the HIV virus. Establishing an effective HIV infection, in a murine model would require the assimilation of human Factor H.

[0045] A variety of sialic acids (characterized by a 9 carbon backbone) and/or a glycan chain (composed of mostly 5 and 6 carbon sugars) are expressed on the surfaces of animals, fungi, plants, protozoa, bacteria and viruses. Mammals possess a variety of sialic acid recognizing proteins known as Siglecs. To date, eleven functional Siglecs and one Siglecs like molecule (Siglec L1) have been characterized. Macrophages express Siglec 1 (sialoadhesin), B cells express Siglec 2 (CD22) and monocytes express Siglec 3 (CD33). Cells involved in the innate immune response including natural killer cells and granulocytes are characterized by Siglecs 1 , 3, 5, 7 and 10. The function of a protein and its potential immunogenicity are in part related to its surface glycan or sialic acid residues. Therefore, a potential rejection and function issue exists if proteins from animals expressing different surface sugar molecules coexist in the same animal. Interestingly, the CMP-Neu5Ac synthetase genes that encode the enzymatic machinery necessary for sialic acids are found with one exception only in fruit flies, rainbow trout, mice and humans. Surprisingly, one bacteria Streptomyces coelicolor also expresses this genetic machinery. Lateral gene transfer between this bacterium and a eukaryotic host best explains this anomaly.²⁴ Therefore, a murine model obviates this overwhelming concern.

[0046] The mucosa of the murine Gl tract has been well described. The surface of Peyer"s patches is covered by epithelium associated with a variety of lymphoid cells known as the follicle-associated epithelium (FAE). The FAE is composed of a variety of cells including cells known as M cells. These cells exhibit slender cytoplasmic extensions around lymphoid cells. The basolateral surface of the M cell is deeply invaginated forming a pocket that shortens the distance from the apical to the basolateral surface. The pocket is rich in B cells, T cells, macrophages and dendritic cells. Antigen uptake by M cells does not result in intracellular degradation but rather delivery of the intact molecule to the underlying lymphoid tissue. The apical surface of the M cell lacks the brush border of typical gut lining enterocytes. Furthermore, the M cells are not coated with the thick glycocalyx found on enterocytes. Finally, the distribution of actin-associated protein villin in M cells differs from enterocytes. These characteristics make M cells ideal targets for absorption of proteins produced by recombinant commensal organisms needed for HIV replication.²⁵

[0047] A variety of methods will target the M cells for absorption of defined proteins. These include, but are not limited to: (1 ) cholera toxin-B subunit, (2) carbohydrate lectins, (3) genetically engineered IgA or the secretory component of IgA. Splicing the genetic DNA sequence for a defined protein needed for HIV replication and linking that protein to 1 , 2 or 3, above, will target the protein to the M cells and ultimately to the underlying immune tissue.²⁶

[0048] Alternatively, attenuated viruses particularly the mouse reovirus, attenuated Poliovirus type 1 and the attenuated Sabin strain selectively adhere to M cells. These viruses can be exploited for transporting a defined protein into Peyer"s patches.²⁷ [0049] Certain attenuated bacteria also target the M cell apical membrane. These include Vibrio Cholerae, Salmonella, Shigella, Yersinia and BCG. Attenuation of these organisms renders them non-virulent. They can be exploited in targeting recombinant proteins to the M cells and the underlying immune tissue.²⁸

[0050] As a final step, the described proteins are administered to the animal by way of its GALT followed by infection of live HIV. Infection with live HIV will result in Tat protein transcription and translation with the resulting Tat mediated immune suppression. Alternatively, Tat protein or the incorporation of the DNA encoding the Tat protein can be administered directly in combination with other proteins or incorporated into the commensal through recombinant technology described above.

Administration and Supplements

[0051] It is possible for the proteins, composition of proteins and or compositions of incorporated DNA encoding the proteins to be administered as a pharmaceutical formulation or preparation, optionally with supplements or other compositions as described above. If protein carriers are used they must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The coupling of protein carriers (e.g., complement proteins) is known within pharmacology.

[0052] Administration may be made in a variety of routes, for example orally, transbucally, transmucosally, sublingually, nasally, rectally, vaginally, intraocularly, intramuscularly, intralymphatically, intravenously, subcutaneously, transdermal^, intradermally, intra tumor, topically, transpulmonarily, by inhalation, by injection, or by implantation, etc. Various forms of the composition may include, without limitation, capsule, gel cap, tablet, enteric capsule, encapsulated particle, powder, suppository, injection, ointment, cream, implant, patch, liquid, inhalant, or spray, systemic, topical, or other oral media, solutions, suspensions, infusion, etc. Because some of the first targets for infection with HIV are epithelial cells and Langerhans cells in the skin and rectal mucosa, then a preferable embodiment of delivery is dermal combined with rectal suppositories.

[0053] Those skilled in the art will recognize that for administration by injection, formulation in aqueous solutions, such as Ringer's solution or a saline buffer may be appropriate. Liposomes, emulsions, and solvents are other examples of delivery vehicles. Oral administration would require carriers suitable for capsules, tablets, liquids, pills, etc, such as sucrose, cellulose, etc.

[0054] The preferred method of administration would be via commensal organisms genetically modified to express one or more human derived proteins needed for HIV replication. The preferred area of administration would be the intestines targeting Peyei^s patches. The delivery and deliberate infection of live HIV is well known in the art and includes intra vaginal, rectal and systemic portals.

[0055] In conclusion, the present invention provides compositions and a method for producing an animal model for HIV induced disease. The present invention is an animal adapted to simulate a human-like immune response to HIV, which is accomplished by activation and inactivation of complement of proteins within the animal. Accordingly, the present invention stages certain human proteins within an animal by way of its GALT followed by infection of live HIV.

[0056] The analysis and development of the animal model for HIV induced disease should incorporate a wide range of doses of the proteins necessary for viral replication and immune evasion, deregulation and/or suppression for evaluation. Animal trials should consider differences in size, species, and immunological characteristics. [0057] The above examples should be considered to be exemplary embodiments, and are in no way limiting of the present invention. Thus, while the description above refers to particular embodiments, it will be understood that many modifications may be made without departing from the spirit thereof.

[0058] Prokaryotic organisms lack the post translational modification machinery found in eukaryotic organisms. Yeast such as Saccharomyces cerevisiae are eukaryotes often found as commensal organisms in GALT. Yeast may therefore be preferable as recombinatorial vectors.

[0059] A blend of genetic manipulations may yield the optimal animal model. A mouse with one or the other above-mentioned amino acid substitutions in the Cyclin T protein that renders it Tat and TAR processive would be a good starting point. This murine model could then assimilate the CD4 receptor and the CCR5 and CXCR4 co- receptors by transgenic technology. Other proteins the mouse is lacking to affect HIV replication, immune evasion and immune disregulation could be supplied in trans via recombinatorial GALT vectors.

References

1. Lindblad-Toh, et al., 2001 , http://www.ncbi.nlm.nih.gov 2. Nagy, Andras, et al., "Manipulating the Mouse Embryo," A Laboratory Manual, 2003, 3d ed., Ch. 1, pp. 1-29

3. Bieniasz , Paul D., et. al., "Analysis of the Effect of Natural Sequence Variation in Tat and in Cyclin T on the Formation and RNA Binding Properties of Tat-Cyclin T Complexes," J [of Virology ; July 1999, Vol. 73, pp. 5777-5786

4. Czerkinsky, Cecil, et. al., "Mucosal immunity and tolerance: relevance to vaccine development," Immunologic Reviews, 1999, Vol. 170, pp. 197-222

5. Cerkinsky, Cecil, et. al., "Mucosal immunity and tolerance: relevance to vaccine development," Immunologic Reviews, 1999, Vol. 170, pp. 197-222

6. Czerkinsky, Cecil, et. al., "Mucosal immunity and tolerance: relevance to vaccine development," Immunologic Reviews, 1999, Vol. 170, pp. 197-222

7. Czerkinsky, Cecil, et. al., "Mucosal immunity and tolerance: relevance to vaccine development," Immunologic Reviews, 1999, Vol. 170, pp. 197-222

8. Huang, Fang-Ping, et. al., "A Discrete Subpopulation of Dendritic Cells Transports Apoptotic Intestinal Epithelial Cells to T Cell Areas of Mesenteric Lymph Nodes," J. Exp. Med., 2000, Vol. 191 , No. 3, Feb 7, pp. 435-443

9. Nicholl, Desmond S. T., An Introduction to Genetic Engineering, 2002, 2d ed., Ch. 4, pp. 43-53

10. Nicholl, Desmond S. T., An Introduction to Genetic Engineering, 2002, 2d ed., Ch. 5, pp. 57-85 11. Nicholl, Desmond S. T., An Introduction to Genetic Engineering, 2002, 2d ed., Ch. 5, pp. 57-85

12. Nicholl, Desmond S. T., An Introduction to Genetic Engineering, 2002, 2d ed., Ch. 5, pp. 57-85

13. Nicholl, Desmond S. T., An Introduction to Genetic Engineering, 2002, 2d ed., Ch. 5, pp. 57-85

14. Nicholl, Desmond S. T., An Introduction to Genetic Engineering, 2002, 2d ed., Ch. 5, pp. 57-85

15. Langel, UIo, Handbook of Cell-Penetrating Peptides, 2007, 2d ed., Ch. 1 , pp. 1- 28

16. Sayah, David. M., et. al., "Cyclophilin A retrotransposition into TRIM5 explain owl monkey resistance to HIV-1 ," Nature, 2004, Vol. 430, July 29^th, pp 569-573

17. Mariani, Roberto, et al., "A Block to Human Immunodeficiency Virus Type 1 Assembly in Murine Cells," Journal of Virology, Vol. 74, No. 8, April 2000, pp. 3859- 3870

18. Bieniasz, Paul D., et al., "Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species specificity of HIV-1 Tat," The EMBO Journal, Vol. 17, 1998, pp. 7056-7065

19. Spear, G. T., et al., "Human immunodeficiency virus (HIV)-infected cells and free virus directly activate the classical complement pathway in rabbit, mouse and guinea- pig sera; activation results in virus neutralization by virolysis," Immunology, Vol. 73, 1991 , pp. 377-382

20. Stoiber, Heribert, et al., "Interaction of several complement proteins with gp120 and gp41, the two envelope glycoproteins of HIV-1 ," AIDS, Vol. 9, 1995, pp. 19-26

21. Stoiber, Heribert, et al., "Efficient Destruction of Human Immunodeficiency Virus in Human Serum by Inhibiting the Protective Action of Complement Factor H and Decay Accelerating Factor (DAF, CD55)," J. Exp. Med, January 1996, Vol. 183, pp. 307-310

22. Kristensen, Torsten, et al., "Murine protein H is comprised of 20 repeating units, 61 amino acids in length," Proc. Nat. Acad. ScL, USA, Vol. 83. June 1986, pp. 3963- 3967

23. Ripoche, Jean, et al., "The complete amino acid sequence of human complement factor H," Biochem. J., Vol. 249, 1988, pp. 593-602

24. Angata, Takashi, et al., "Chemical Diversity in the Sialic Acids and Related α- Keto Acids: An Evolutionary Perspective," Chem. Rev., Vol. 102, 2002, pp. 439-469

25. Kiyono, Horishi, et al., Mucosal Vaccines, 1996, Ch. 2, pp. 17-40 and Ch. 3, pp. 41-53

26. Kiyono, Horishi, et al., Mucosal Vaccines, 1996, Ch. 3, pp. 41-53

27. Kiyono, Horishi, et al., Mucosal Vaccines, 1996, Ch. 3, pp. 41-53

28. Kiyono, Horishi, et al., Mucosal Vaccines, 1996, Ch. 3, pp. 41-53 29. S., Bounou, et al., The importance of virus-associated host ICAM-1 in human immunodeficiency virus type 1 dissemination depends on the cellular context, FASEB J. 2004 Aug;18(11):1294-6. Epub 2004 Jun 18

Appendix A

The following information is generally known by those in the art and can be found in medical texts generally including by way of example , Mucosal Vaccines. Hematology Basic Principles and Practices, and Immunology. Infection and Immunity and journals such as Immunologic Reviews, Nature, Virology, and Molecular Immunology.

regulates expression of MHC class Il genes in antigen-presenting cells (APC) by inhibiting the transactivator of MHC class Il genes, CIITA. HIV-1 Tat up regulates HLA-DR expression in monocyte-derived dendritic cells and T cells, thereby driving T cell-mediated immune responses and activation. Associates with HIV-1 gp41. Enhances HIV-1 infectivity. Not affected by viral tropism which is determined by the V3 loop of gp120. Amino acids 708-750 of gp41 required for MHC-II incorporation into the HIV-1 envelope.

Approximately 375 to 600 molecules of HLA Il are incorporated into each HIV-1 virion. HLA Il DR is the predominant if not only subtype of HLA Il detected on the surface of most HIV-1 virions. Therefore, HLA Il DR is selectively incorporated into the viral envelope.

ICAM-1 A type 1 transmembrane Yes, envelope Increases HIV macrophages. HIV-1 group N and group O Nef weakly down regulates CD4, CD28, and class I and Il MHC molecules and up regulates surface expression of the invariant chain (Ii) associated with immature major histocompatibility complex (MHC) class II. Nef interrupts MHC- I trafficking to the plasma membrane and inhibits antigen presentation. Nef interacts with the μ1 subunit of adaptor protein (AP) AP-1A, a cellular protein complex implicated in TGN linking endosome/lysosome pathways. HIV-1 Nef binds to the MHC-I (HLA-A2) hypo phosphorylated cytoplasmic tails in the endoplasmic reticulum; this Nef-MHC-I complex migrates into the Golgi apparatus then into the lysosomal compartments for degradation. Nef promotes a physical interaction between endogenous AP- 1 and MHC-I. The Pro-X-X- Pro motif in HIV-1 Nef induces the accumulation of CCR5 (HIV-1 M-tropic coreceptor) in a perinuclear compartment where both molecules co- localize with MHC-1. The Pro-X-X-Pro motif interacts with src homology region-3 domains of src-like kinases interfering with cell signaling pathways. HIV-1 Nef selectively down regulates HLA-A and HLA-B but does not significantly affect HLA-C or HLA-E₁ which allows HIV- infected cells to avoid NK cell-mediated lysis. Nef decreases the incorporation of MHC-1 molecules into virions. Furthermore, Nef down regulates MHC-1 expression on human dendritic cells. Therefore, HIV-1 Nef impairs antigen presentation to HIV- specific CD8+ T lymphocytes. HIV-1 Nef-induced down regulation of MHC-I expression and MHC-I targeting to the trans- Golgi network (TGN) require the binding of Nef to PACS-1 (phosphofurin acidic cluster sorting protein 1). PACS-1 is a protein with a putative role in the localization of proteins to the trans- Golgi network (TGN) including furin which cleaves gp160. HIV-1 Nef down regulates MHC-1 on lymphoid, monocytic and epithelial cells. Nef expression results in rapid internalization and accumulation of MHC-1 in endosomal vesicles which degrade MHC-1 molecules. Nef blocks transport of MHC-I molecules to the cell surface, leading to accumulation of MHC- 1 in intracellular organelles.

Furthermore, the effect of Nef on MHC-1 molecules (but not on CD4) requires phosphoinositide 3- kinase (Pl 3-kinase) activity found on the cytoplasmic side of the plasma membrane.

CD63 A type III Yes, envelope The efferent arm of transmembrane protein viral replication occurs present on activated in the endosomes. platelets, monocytes, The CD63 marker is phosphatase facilitates dephosphorylation of phosphorylated Cdc25 protein by the protein phosphatase PP2A. Found on chromosome 19 location 19p13. Mediates GM-CSF production. Binds c-Fos through specific pS/T-P sites within the c-Fos TAD (carboxyl terminal transactivation domain) resulting in enhanced transcriptional response of c-Fos to polypeptide growth factors that stimulate ERK (extracellular regulated kinases). Involved in the cooperative activity of c- Jun and c-Fos to regulate AP-1 - dependent gene transcription upon phosphorylation by mitogen-activated kinase (MAPK) family members. Binds to the pThr254- Pro motif in p65 and inhibits p65 binding to l_κBα, resulting in increased nuclear accumulation and protein stability of p65 and enhanced NF_KB activity. Interacts with transcription factor β- catenin (cadherin- associated protein) and increases the variants have been characterized.

CypA Immunophilin, peptidyl- Yes, virion Incorporated as a (Cyclophilin A) prolyl isomerase. Found component of the Gag on chromosome 7 molecule at a 1/10 location 7p13. ratio. Also interacts Catalyzes the cis-trans with Vpr, Vif, MA, Nef isomerization of proline and gp120env. Binds imidic peptide bonds in to the central region of oligopeptides, the CA protein accelerates the folding of (residues 85 to 93). proteins. Catalyzes the cis/trans isomerization of the Gly-89-Pro-90 peptide bond. The capsid sequence 87HiS-AIa- Gly-Pro-lle-Ala92 (87HAGPIA92) encompasses the primary cyclophilin A binding site. Inhibits ltk (lnterieukin-2 tyrosine kinase) catalytic activity, a cytoplasmic nonreceptor protein tyrosine kinase of the Tec (Molecular class: tyrosine kinase, Molecular Function: protein-tyrosine kinase activity, Biological Process: cell communication, signal transduction) family that participates in the intracellular signaling events leading to T cell activation. A proline- dependent conformational switch within the ltk SH2 domain regulates substrate recognition and mediates regulatory interactions with the active site of CypA. Regulates the cis/trans interconversion of the imidic bond within the conserved proline residues of Vpr in vivo. Implicated in capsid final assembly and defense of HIV-1 against innate host restriction factors specifically Ref-1. CypA/CD147 (Type l integral membrane glycoprotein found on hemopoietic, microglial, endothelial and peripheral blood cells) interaction follows CypA interaction with surface heparins. Facilitates viral/host cell binding prior to gp120/CD4 and gp120/CXCR4 or CCR5 co-receptor interaction. Increases probability of successful infection when a small amount of virus has been transmitted.

FKBP12 (FK506 A peptidyl prolyl Yes, virion Growth of chronically binding proteins) isomerase. Found on infected HIV-1 cells chromosome 20 location dependent on FKBP12

UNG (Uracil- Uracil-DNA glycosylase Yes, virion lntegrase is required

DNA removes DNA uracil for packaging of UNG glycosylase) residues. Excises the into virions. UNG2 uracil residues and binds the viral reverse introduces non transcriptase enzyme. templated nucleotides Uracil repair pathway allowing for somatic is associated with HIV- hyper mutation. 1 viral particles. Increases immunoglobulin diversity. Essential for generation of strand breaks for class switch recombination. Both mitochondrial (UNG1 ) and nuclear (UNG2) isoforms have been described. UNG1 only uracil-DNA glycosylase isolated to date in mitochondria. Mitochondrial UNG1 is encoded by nuclear not mitochondrial DNA. UNG2 predominant form in proliferating cells, UNG1 predominant form in non-proliferating cells. UNG2 levels high in S- phase and early G2 of the cell cycle. UNG2 primarily located at replication foci during S- phase. A second uracil- DNA glycosylase, Single-strand-selective Monofunctional Uracil- DNA Glycosylase (SMUGI) has a preference for double- stranded DNA rather responsive element p300 for CDK9/P-TEFb binding protein]). Like CTD kinase complex. CBP can stimulate Tat binds to amino acid transcription through 1253-1790 of p300. activation of CREB. This interaction results in a structural change of p300/CBP. Tat-p300 interaction increases the HAT activity of p300 on histone H4. H4 is a component of nucleosomes. Histone H4 was acetylated on lysines 8, 12, and 16. Acetylation of H4 was inhibited by Lys- coenzyme A (CoA), a selective inhibitor of p300 acetyltransferase activity. Tat could auto acetylate itself, which was specific to lysine residues 41 and 71. Acetylated Tat is considered to be the transcriptionally active form intracellularly. p300 and PCAF directly acetylate Tat. p300 acetylated Lys50 in the TAR RNA binding domain, while PCAF acetylated Lys28 in the activation domain of Tat. Acetylation at Lys28 by PCAF enhanced Tat binding to the Tat- associated kinase, CDK9/P-TEFb, while acetylation by p300 at

HLA-DR Antigen presentation, Yes, envelope Interacts with CD4 MHC class Il directly glycoprotein on target presents peptide cells. Without antigens to CD4 T cells. associated antigen in Highly polymorphic. the peptide binding Heterodimer consisting cleft of HLA-DR and of an alpha (DRA) and a co-stimulating beta (DRB) chain, both molecular interactions, anchored in the CD4 cell will be membrane. Presents rendered anergic. peptides derived from HIV-1 Gag expression extracellular proteins by is able to induce HLA- antigen presenting ceils, DR cell-surface B cells, dendritic cells localization in H78- and macrophages. C10.0 cells. In human Found on chromosome macrophages, HIV-1 6 location 6p21.3. Gag proteins co- localize with MHC Il (HLA-DR), CD63, and Lampi in MHC Il compartments. HIV-1 Capsid (p24) inhibits interferon gamma induced increases in HLA-DR and cytochrome B heavy chain mRNA levels in the human monocyte- like cell line THP1. HIV-1 Tat down regulates expression of MHC class Il genes in antigen-presenting cells (APC) by inhibiting the transactivator of MHC class Il genes, CIITA. HIV-1 Tat up regulates HLA-DR expression in monocyte-derived dendritic cells and T cells, thereby driving T cell-mediated immune responses and activation. Associates with HIV-1 gp41. Enhances HIV-1 infectivity. Not affected by viral tropism which is determined by the V3 Ioop of gp120. Amino acids 708-750 of gp41 required for MHC-II incorporation into the HIV-1 envelope.

MHC-1 In humans, six MHC Yes, envelope Enhances HIV class 1 isotypes have infectivity and changes been identified: HLA-A, gp120 conformation. HLA-B₁ HLA-C₁ HLA-E, Without antigen in HLA-F and HLA-G. MHC-1 binding groove HLA-A₁ HLA-B and HLA- and co-stimulatory C function to present activity, anergy results. antigens to CD8 T cells HIV-1 Nef down and to form ligands for regulates surface natural killer (NK) cell expression of CD4 and receptors. HLA-E and MHC-1 in resting CD4⁺ HLA-G also ligands for T lymphocytes. Nef NK-cell receptors. HLA- up regulates cell A is found on surface levels of the chromosome 6 location MHC-2 invariant chain

6p21.3. CD74. Nef down regulates HLA class I expression and therefore suppresses the cytolytic activity of HI V-1 -specific cytotoxic T-lymphocyte (CTL) clones. Macrophage-tropic (M- tropic) HIV-1 Nef down regulates expression of HLA-A2 on the surface of productively infected macrophages. HIV-1 group N and group O Nef weakly down regulates CD4, CD28, and class I and Il MHC molecules and up regulates surface expression of the invariant chain (Ii) associated with immature major histocompatibility complex (MHC) class II. Nef interrupts MHC-I trafficking to the plasma membrane and inhibits antigen presentation. Nef interacts with the μ1 subunit of adaptor protein (AP) AP-IA, a cellular protein complex implicated in TGN linking endosome/lysosome pathways. HIV-1 Nef binds to the MHC-I (HLA-A2) hypo phosphorylated cytoplasmic tails in the endoplasmic reticulum; this Nef- MHC-I complex migrates into the Golgi apparatus then into the lysosomal compartments for degradation. Nef promotes a physical interaction between endogenous AP- 1 and MHC-I. The Pro-X-X- Pro motif in HIV-1 Nef induces the accumulation of CCR5 (HIV-1 M-tropic coreceptor) in a perinuclear compartment where both molecules co- localize with MHC-1. The Pro-X-X-Pro motif interacts with src homology region-3 domains of src-like kinases interfering with cell signaling pathways. HIV-1 Nef selectively down regulates HLA-A and HLA-B but does not significantly affect HLA-C or HLA-E, which allows HIV- infected cells to avoid NK cell-mediated lysis. Nef decreases the incorporation of MHC- 1 molecules into virions. Furthermore, Nef down regulates MHC-1 expression on human dendritic cells. Therefore, HIV-1 Nef impairs antigen presentation to HIV- specific CD8+ T lymphocytes. HIV-1 Nef-induced down regulation of MHC-I expression and MHC-I targeting to the trans- Golgi network (TGN) require the binding of Nef to PACS-1 (phosphofurin acidic cluster sorting protein 1). PACS-1 is a protein with a putative role in the localization of proteins to the trans-Golgi network (TGN) including furin which cleaves gp160. HIV-1 Nef down regulates MHC-1 on lymphoid, monocytic and epithelial cells. Nef expression results in rapid internalization and accumulation of MHC-1 in endosomal vesicles which degrade MHC-1 molecules. Nef blocks transport of MHC-I molecules to the cell surface, leading to accumulation of MHC- 1 in intracellular organelles.

Furthermore, the effect of Nef on MHC-1 molecules (but not on CD4) requires phosphoinositide 3- kinase (Pl 3-kinase) activity found on the cytoplasmic side of the plasma membrane.

HSP70 (Heat Chaperone intracellular Yes, virion May bind HIV-1 gag shock protein protein produced in polyprotein chain and 70) response to intracellular maintain proper stress. Found on tertiary confirmation chromosome 19 location during intracellular

19q13.42. Binds to and transport from nucleus regulates Hsp70 activity. to plasma membrane.

The carboxyl terminus of May participate in

Hsp70-interacting early events in protein (CHIP) is an infection. Might

Hsp70-associated participate in ubiquitin ligase which uncoating the viral ubiquitinates misfolded capsid. May target proteins associated with HIV-1 PIC to the cytoplasmic nucleus. chaperones.

UNG (Uracil- Uracil-DNA glycosylase Yes, virion lntegrase is required

DNA removes DNA uracil for packaging of UNG glycosylase) residues. Excises the into virions. UNG2 uracil residues and binds the viral reverse introduces non transcriptase enzyme. templated nucleotides Uracil repair pathway allowing for somatic is associated with HIV- hyper mutation. 1 viral particles.

Increases immunoglobulin diversity. Essential for generation of strand breaks for class switch recombination. Both mitochondrial (UNG1 ) and nuclear (UNG2) isoforms have been described. UNG1 only uracil-DNA glycosylase isolated to date in mitochondria. Mitochondrial UNG1 is encoded by nuclear not mitochondrial DNA. UNG2 predominant form in proliferating cells, UNG1 predominant form in non-proliferating cells. UNG2 levels high in S- phase and early G2 of the cell cycle. UNG2 primarily located at replication foci during S- phase. A second uracil- DNA glycosylase, Sing le-strand-selecti ve Monofunctional Uracil- DNA Glycosylase (SMUGI) has a preference for double- stranded DNA rather than single-stranded DNA as with UNG1 and UNG2. Found on chromosome 12 location 12q23-q24.1. Not cell cycle regulated, does not accumulate at replication fosi and is not found in mitochondria. SMUG1 accumulates in nucleoli, UNG2 excluded from nucleoli. UNG1. UNG2 and SMUG1 function in base excision repair. UNG2 implicated in both innate and acquired immunity.

Staufen Double-stranded RNA Yes, virion Binds HIV-1 genomic binding protein. RNA. May be involved Transports mRNAs to in retroviral genome intracellular selection and compartments/organelle packaging into s. Found on assembling virions. chromosome 20 location Interaction with the 20q13.1. Binds tubulin. nucleocapsid domain Transports mRNA via of pr55(Gag) in vitro the microtubule network and in live cells to the RER. Five mediated by Staufen's transcript variants from dsRBD3 (RNA binding alternative splicing of domain 3), with a STAU gene encoding contribution from its C- three isoforms have terminal domain. been described. Preferentially binds with the 9-kb non- spliced viral RNA. Implicated in the generation of infectious virions. α-actinin 1 Required for Vpx- mediated nuclear import of the PIC.

LEDGF/p75 DNA-binding protein Yes, PIC Central core domain (lens epithelium- implicated in cellular (preintegration and N-terminal zinc derived growth differentiation and complex) binding domain of factor/transcripti cellular response to integrase are involved on coactivator environmental stress. in the interaction with p75 [alternate Activates transcription of LEDGF/p75. An names include stress related genes essential cofactor for PC( positive co- triggering a survival nuclear targeting of factor^ and response. Protective HIV-1 integrase. SFRS1 role in stress-induced Physically links interaction apoptosis. Found on integrase to host protein 2 chromosome 9 location chromatin. The (PSI P2)]) Prior 9p22.3. A member of alternatively spliced journal articles the hepatoma-derived protein LEDGF/p52, differentiate p75 growth factor (HDGF). does not interact with from PC4 in The alternatively spliced HIV-1 or HIV-2 HELA cells p52 (PC4 and SFRS1 integrase. interaction protein 1 LEDGF/p75 links the (PSIP1 )) protein integrase protein to interactions with the host chromatin transcriptional during the G₂ phase of coactivators, general the cell cycle. May transcription factors, and target the HIV-1 splicing factors, proviral DNA to modulating pre-mRNA specific genomic sites splicing of class Il of actively transcribed genes. The p75 protein genes to promote viral is not a transcriptional transcription. factor. Heparin binds to Residues are the LEDGF/p75, facilitating integrase binding transport through the domain (IBD). cytoplasm into the Dictates site(s) of HIV nucleus. The N-terminal integration, most PWWP domain and its favored are areas beta-barrel substructure undergoing are needed for binding transcription, high G-C to metaphase (guanine-cytosine) chromatin. content, with active RNA polymerase subunits and transcription factors. Prevents proteasomal degradation of HIV-1 integrase. The N- terminal zinc binding domain (amino acids 1-52) and the central core domain (amino acids 53-212) of HIV-1 integrase interact with LEDGF/p75. The core domain harbors the main determinant for this interaction. tRNA synthetase Ligase, charges or Yes, virion tRNA^lys3 binds to the or aminoacyl aminoacylates key RNA primer binding site tRNA synthetase molecules linking the initiating reverse molecule to the transcription. In HIV-1 respective amino acid. an RNA loop formed One synthetase for each by the tRNA^lys3 amino acid found in anticodon and an mammalian cells. ATP adenine rich RNA loop dependent. initiates reverse transcription. tRNA^lys Allows incorporation of Yes, virion Induces three lysine into proteins by associated dimensional structural the host translational attached to changes in the apparatus. primer binding unspliced viral RNA to site (PBS) allow reverse transcription to proceed.

GAPDH In glycolysis, Yes, virion 979999

(Glyceraldehyde enzymatically converts -3-phosphate Glyceraldehyde-3- dehydrogenase) phosphate to 1 , 3- 6/sphosphoglycerate. Also involved in cell cycle regulation by modulating cyclin B- cdk1, apoptosis, membrane fusion, microtubule bundling, phosphotransferase activity, nuclear RNA export, programmed neuronal cell death, DNA replication, and DNA repair. Found on chromosome 12 location 12p13.

CD4 A type I transmembrane Yes, envelope Interacts with specific protein found on domains of gp120 helper/inducer T cells, facilitating viral fusion. monocytes, macrophages, and dendritic cells that is involved in T-cell recognition of antigens. Found on chromosome 12 location 12pter-p12.

CXCR4 Binds chemokine SDF-1 Yes, envelope Viral co-receptor (stromal cell derived determines viral factor 1 ). Found on tropism for CD4 T hematopoietic cells. precursors, mature white blood cells and plasma cells. Found on chromosome 2 location 2q21. Type fll transmembrane protein crossing the plasma membrane seven times.

CCR5 Found on Th1 cells, Yes, envelope Viral co-receptor dendritic cells, determines viral monocytes/macrophage tropism for s. Type III macrophages. transmembrane protein crossing the plasma membrane seven times. Ligands include monocyte chemo attractant protein 2 (MCP-2), macrophage inflammatory protein 1 alpha (MIP-1 alpha), macrophage inflammatory protein 1 beta (MIP-1 beta) and regulated on activation normal T expressed and secreted protein (RANTES). Found on chromosome 3 location 3p21.31

NF_KB Cellular transcription Binding sites in the factor involved in the viral LTR necessary immune process. for viral transcription.

Found on chromosome * location *. NFAT Cellular transcription Binding sites in the factor involved in the viral LTR necessary immune process. for viral transcription.

Found on chromosome

20 location 20q13.2- q13.3. Sp1 Cellular transcription Binding sites in the factor involved in the viral LTR necessary immune process. for viral transcription.

Found on chromosome

12 location 12q13.1.

Claims

CLAIMSWhat is claimed is:

1. A method for the production of an animal model for HIV comprising the steps of: a. creating and administering HIV related proteins necessary for HIV to attach, penetrate, and replicate within a live animal by encoding said proteins into commensal organisms derived from gut associated lymphoid tissue using recombinant technology; b. creating and administering HIV related proteins necessary for HIV to evade said animal's immune response by encoding said HIV related proteins into commensal organisms derived from gut associated lymphoid tissue using recombinant technology; and c. infecting said animal with live, replication competent HIV.

2. The method of claim 1, wherein said HIV related protein concentrations administered to said animal are supplied in trans and mirror concentrations found in normal human immunologic milieu.

3. The method of claim 1, wherein said method further comprises the step of

coupling said HIV related proteins with cell penetrating peptides using recombinant technology.

4. The method of claim 1, wherein said method further comprises the step of administering CypA-binding drug Cyclosporine to said live animal.

5. The method of claim 1, wherein said method further comprises the step of administering soluble complement-receptor 1 to said live animal.

6. The method of claim 1, wherein said method further comprises the step of administering Tat protein to said live animal.

7. The method of claim 1, wherein the HIV related proteins necessary for HIV to attach, penetrate, and replicate within said live animal are selected from transcription factors, cellular factors, cellular receptors, cellular co-receptors, cellular proteases, cellular proteins involved in the ubiquitin-proteasome pathway, cellular adaptor proteins, human ribosomal RNA, and combinations thereof.

8. The method of claim 1, wherein the HIV related proteins necessary for HIV to attach, penetrate, and replicate within said live animal include transcription factors and the transcription factors are selected from NF_KB, NFAT, SpI, and

combinations thereof.

9. The method of claim 1, wherein the HIV related proteins necessary for HIV to attach, penetrate, and replicate within said live animal include cellular cofactors and the cellular cofactors are selected from Cyclin T, CDK9/PITALRE, RNA polymerase II, Exportin 1/Cπnl, Ran GTP, Ran GTPase activating protein (RanGAP), Ran Binding Protein (RanBPl), and combinations thereof.

10. The method of claim 1, wherein the HIV related proteins necessary for HIV to attach, penetrate, and replicate within said live animal include cellular receptors and the cellular receptors are CD4.

11. The method of claim 1, wherein the HIV related proteins, necessary for HIV to attach, penetrate, and replicate within said live animal include cellular coreceptors and the cellular coreceptors are selected from CCR5, CXCR4, CCR2B, CCR3, CCR8, GPRl, GPRl 5 (Bob), STRL33 (Bonzo), US28, CX3CR1 (V28), APJ, chemR23, and combinations thereof.

12. The method of claim 1, wherein the HIV related proteins necessary for HIV to attach, penetrate, and replicate within said live animal include cellular proteases and the cellular protease is Furin.

13. The method of claim 1, wherein the HIV related proteins necessary for HIV to attach, penetrate, and replicate within said live animal include cellular proteins involved in the ubiquitin-proteosome pathway and the cellular proteins involved in the ubiquitin-proteosome pathway are selected from H-β-TrCP, Skplp, and combinations thereof.

14. The method of claim 1, wherein the HIV related proteins necessary for HIV to attach, penetrate, and replicate within said live animal include cellular adaptor proteins and the cellular adaptor proteins are AP-2.

15. The method of claim 1, wherein the HIV related proteins necessary for HIV to evade said animal's immune response are selected from plasma proteins, cell membrane bound proteins, homologous restriction factor (HRF), said animal

proteins incorporated into the intact virus, said animal proteins incorporated into the pre-integration complex (PIC), and combinations thereof.

16. The method of claim 1, wherein the HIV related proteins necessary for HIV to evade said animal's immune response include plasma proteins and the plasma proteins are selected from C4 binding protein (C4b protein), factor H and combinations thereof.

17. The method of claim 1, wherein the HIV related proteins necessary for HIV to evade said animal's immune response include cell membrane bound proteins and the cell membrane bound proteins are selected from membrane cofactor protein (MCP) or CD46, decay accelerating factor (CD55), complement-receptor 1 (CD35), complement-receptor 2 (CD21), homologous restriction factor, and combinations thereof.

18. The method of claim 1, wherein said HIV related proteins necessary for HIV to evade said animal's immune response include said animal's proteins and said animal's proteins are selected from the HIV-I related proteins that are selected from MCP/CD46, DAF/CD55, HRF-20/CD59, Factor H, Thy-1 (CD90), GMl (β- galactosidase), HLA-DR, ICAM-I, ICAM-2, ICAM-3, LFA-I, VCAM-I, VLA-4, MHC-I, CD63, CD81, CD82, CD 107a, HP68, ezrin, moesin, cofilin, actin, ubiquitin, Pinl, tRNA synthetase, aminoacyl tRNA synthetase, GAPDH, MAPK/ERK2, HSP60, HSP70, HSC70, CypA, FKBP12, TsglOl, TaI, VPS28, AIP1/ALIX,VPS4B, APOBEC3G, APOBEC3F, UNG, Staufen, INIl, EF- lα, LEDGF/p75, PSIP2, DNA-PK, Ku80, hRadl8, EED, HMGA/HMG-la, BAF/BANF1, p300, Rev cofactor, HSp90, CypB, HSP 27, HSP40, VPS37B, CD4, CXCR4, CCR5, CD86, Phosphatidyl inositol 4,5-bisphosphate, NF_KB, NFAT, SpI, Cyclin T, CDK9/PITALRE, RNA polymerase II, Exportiπ 1/Crm 1, Ran GTP, Ran GTPase activating protein, Ran Binding Protein, CCR2B, CCR3, CCR8, GPRl, GPRl 5, STRL33, US28, CX3CR1, APJ, chemR23, Furin, H-β- TrCP, Skplp, AP-2, C4 binding, protein, CD35, CD21, and combinations thereof.

19. The method of claim 1, wherein said HIV related proteins necessary for HIV to evade said animal's immune response include said animal's proteins and said animal's proteins are selected from the HIV-2 related proteins that are selected from HLA-DR, MHC-I, HSP70, UNG, Staufen, α-actinin 1, LEDGF/P75, tRNA synthetase, aminoacyl tRNA synthetase, tRNA^lys, GAPDH, CD4, CXCR4, CCR5, NF_KB, nfat, SpI, and combinations thereof.

20. A composition comprising: a. HIV related proteins necessary for a HIV virion to attach, penetrate, and replicate within a live animal, wherein said proteins are encoded in a genetically engineered commensal organism derived from gut associated lymphoid tissue using recombinant technology; b. HIV related proteins necessary for HIV to evade said animal's immune response, wherein said proteins are encoded in a genetically engineered commensal organism derived from gut associated lymphoid tissue using recombinant technology; and c. live, replication competent HIV.

21. The composition of claim 20, wherein said HIV related proteins are supplied in trans and mirror concentrations found in the normal human immunologic milieu.

22. The composition of claim 20, wherein said HIV related proteins are coupled with DNA encoding a cell penetrating peptide.

23. The composition of claim 20, in combination with CypA-binding drug Cyclosporine.

24. The composition of claim 20, in combination with soluble complement-receptor 1.

25. The composition of claim 20, in combination with Tat protein

26. The composition of claim 20, wherein said HIV related proteins necessary for HIV to attach, penetrate, and replicate within said live animal are selected from transcription factors, cellular factors, cellular receptors, cellular co-receptors, cellular proteases, cellular proteins involved in the ubiquitin-proteasome pathway, cellular adaptor proteins, human ribosomal RNA, and combinations thereof.

27. The composition of claim 20, wherein said HIV related proteins necessary for HIV to attach, penetrate, and replicate within a target cell of said live animal include transcription factors and the transcription factors are selected from NF_KB, NFAT, SpI, and combinations thereof.

28. The composition of claim 20, wherein said HIV related proteins necessary for HIV to attach, penetrate, and replicate within said live animal include cellular cofactors and the cellular cofactors are selected from Cyclin T, CDK9/PITALRE, RNA polymerase II, Exportin 1/Crml, Ran GTP, Ran GTPase activating protein (RanGAP), Ran Binding Protein (RanBPl), and combinations thereof.

29. The composition of claim 20, wherein said HIV related proteins necessary for HIV to attach, penetrate, and replicate within said live animal include cellular receptors and the cellular receptors are CD4.

30. The composition of claim 20, wherein said HIV related proteins necessary for HIV to attach, penetrate, and replicate within said live animal include cellular coreceptors and the cellular coreceptors are selected from CCR5, CXCR4, CCR2B, CCR3, CCR8, GPRl, GPRl 5 (Bob), STRL33 (Bonzo), US28, CX3CR1 (V28), APJ, chemR23, and combinations thereof.

31. The composition of claim 20, wherein said HIV related proteins necessary for HIV to attach, penetrate, and replicate within said live animal include cellular protease and the cellular protease is Furin.

32. The composition of claim 20, wherein said HIV related proteins necessary for HIV to attach, penetrate, and replicate within said live animal include cellular proteins involved in the ubiquitin-proteasome pathway and the cellular proteins involved in the ubiquitin-proteasome pathway are selected from H-β-TrCP, Skplp, and combinations thereof.

33. The composition of claim 20, said HIV related proteins necessary for HIV to attach, penetrate, and replicate within said live animal include cellular adaptor proteins and the cellular adaptor proteins are AP-2.

34. The composition of claim 20, wherein said HIV related proteins necessary for HIV to evade said animal's immune response are selected from plasma proteins, cell membrane bound proteins, homologous restriction factor (HRF), host proteins incorporated into the intact virus, host proteins incorporated into the pre- integration complex (PIC), and combinations thereof.

35. The composition of claim 20, wherein said HIV related proteins necessary for HIV

to evade said animal's immune response include plasma proteins and the plasma proteins are selected from C4 binding protein (C4b protein), factor H, and

combinations thereof.

36. The composition of claim 20, wherein said HIV related proteins necessary for HIV to evade said animal's immune response include cell membrane bound proteins and the cell membrane bound proteins are selected from membrane cofactor protein (MCP) or CD46, decay accelerating factor (CD55), complement-receptor 1 (CD35), complement-receptor 2 (CD21), homologous restriction factor, and combinations thereof.

37. The composition of claim 20, wherein said HIV related proteins necessary for HIV to evade said animal's immune response include said animal's proteins and said animal's proteins are selected from the HIV-I related proteins that are selected from MCP/CD46, DAF/CD55, HRF-20/CD59, Factor H, Thy-1 (CD90), GMl (β- galactosidase), HLA-DR, ICAM-I, ICAM-2, ICAM-3, LFA-I, VCAM-I, VLA-4, MHC-I, CD63, CD81, CD82, CD107a, HP68, ezrin, moesin, cofϊlin, actin, ubiquitin, Pinl, tRNA synthetase, aminoacyl tRNA synthetase, GAPDH, MAPK/ERK2, HSP60, HSP70, HSC70, CypA, FKBP12, TsglOl, TaI, VPS28, AIP1/ALIX,VPS4B, APOBEC3G, APOBEC3F, UNG, Staufen, INIl, EF-lα, LEDGF/p75, PSIP2, DNA-PK, Ku80, hRadlδ, EED, HMGA/HMG-la, BAF/BANF1, p300, Rev cofactor, HSp90, CypB, HSP 27, HSP40, VPS37B, CD4, CXCR4, CCR5, CD86, Phosphatidyl inositol 4,5-bisphosphate, NF_KB, NFAT, SpI, Cyclin T, CDK9/PITALRE, RNA polymerase II, Exportin 1/Cπn 1, Ran GTP, Ran GTPase activating protein, Ran Binding Protein, CCR2B, CCR3, CCR8, GPRl, GPRl 5, STRL33, US28, CX3CR1, APJ, chemR23, Furin, H-β- TrCP, Skplp, A{-2, C4 binding,protein, CD35, CD21, sCRl, and combinations thereof.

38. The composition of claim 20, wherein said HIV related proteins necessary for HIV to evade said animal's immune response include said animal's proteins and said animal's proteins are selected from the HIV-2 related proteins that are selected from HLA-DR, MHC-I, HSP70, UNG, Staufen, α-actinin 1, LEDGF/P75, tRNA synthetase, aminoacyl tRNA synthetase, tRNA^lys, GAPDH, CD4, CXCR4, CCR5, NF_KB, nfat, SpI, and combinations thereof.